Nucleic acid binding species (aptamers) have emerged as a powerful tool for molecular recognition, and have begun to be widely adapted as biosensors, in drug-delivery systems, and as regulatory elements that control gene expression [1]-[4]. Naturally occurring nucleic acid regulatory elements, riboswitches, have been discovered in a variety of organisms and control the expression of a wide range of genes [5].
One of the major advantages of aptamers over their protein counterparts is that they can be easily coupled to other functional RNAs based largely on secondary structural considerations in order to generate allosteric constructs. To a large extent aptamer-based biosensors (both in vitro and in vivo) can be classified into two major categories: (i) those in which the aptamer binding influences the hybridization state of other nucleic acids and (ii) those in which aptamer binding influences the catalysis of a ribozyme. These allosteric ribozymes derived from aptamers are also known as aptazymes.
Aptamers are nucleic acids that bind their cognate ligands (ranging from metal ions to small molecules to proteins) specifically and tightly. Through rational design and/or directed evolution, aptamers can be engineered into allosteric nucleic acids whose conformations can be regulated by their ligands. Aptamer beacons, aptazymes, and riboswitches all undergo ligand-dependent conformational changes, and have been adapted to signal the concentration of their ligands.
Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes, also known as allosteric ribozymes or aptazymes, have been generated by combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme), typically connected to each other via a communication module (single or double strand RNA or DNA).
However, a problem of the highly sensitive aptazyme sensors is that increased sensitivity is accompanied by residual background activity that is detrimental for shelf-life and therefore an obstacle to commercial use.
There is further a need in the art for overcoming the limitation of parallelization of a sensor reaction in a single reaction vessel.
Furthermore, aptamer probes binding unspecifically to surfaces is a widespread problem and reduces sensor performance by leading to false positive results and decreasing sensor sensitivity.
The present disclosure aims to provide improved aptazyme sensors which overcome the limitations of the known aptazyme sensors and which allow sensitive and parallel detection of ligands.
It is a further objective of the present disclosure to provide uses and methods for detecting ligands or analytes in samples as well as for detecting different ligands or analytes in samples.